Several strains of oral streptococci produced fructanase when grown in the absence of D-fructan in a complex medium supplemented with D-glucose. The major part of the activity was extracellular, and only 1-5% was associated with the cells. Release of fructanase began early in the exponential phase and the enzyme was stable in the stationary phase for several h if the pH did not fall below 6. Among the strains of Streptococcus mutans, serotypes a, d, and g released the highest amount of fructanase, and the low level of enzyme produced by strains of serotype c was increased when D-fructose replaced D-glucose as carbon source for growth. Fructanase of S. mutans readily hydrolysed (2 leads to 6)-beta-D-fructans, but (2 leads to 1)-beta-D-fructans and inulin were more resistant. Adsorption of fructanase to (2 leads to 6)-beta-D-fructan, or inhibition with Tris buffer, provided effective means of eliminating fructanase activity from culture filtrates. This procedure should permit a more accurate determination of fructosyltransferase activity of S. mutans strains.